Western Blot Troubleshooting: 8 Protocol Issues Solved + New Tools
Originally published by StressMarq Biosciences June 2015 | Updated July 2025
Western blotting remains a cornerstone of protein analysis, but even seasoned researchers face challenges. We’ve updated our original guide with new tools and insights from 2020–2025 to help you troubleshoot faster and more effectively!
Technical support page: https://www.stressmarq.com/support/technical-support/troubleshooting/western-blot-troubleshooting/
Most of us have had trouble getting a Western blot protocol to work at some point. Don’t get discouraged.
Follow our western blot troubleshooting guide to quickly target the potential cause, and test out solutions.
Common Western Blot Issues and How to Solve Them
- Weak or No Signal
- High Background
- Multiple Bands
- Uneven Band Intensity
- Smiling Bands
- Non-Specific Binding
- Ghost Bands
- Blotchy or Speckled Background
1. Problem: Weak or No Signal
Potential Cause: Incorrect antibody concentration
- Use a higher concentration of antibody, or try incubating longer
- Always optimize both primary and secondary antibodies with every experiment
Potential Cause: Primary antibody does not recognize the antigen
- Check data sheets and reference material to verify that the antibody does detect the antigen in the species
Potential Cause: Primary and secondary antibody mismatch
- The secondary antibody should be raised against the host species of the primary antibody. For example, if the primary was raised in mouse (ie. Mouse Anti-HSP70) use an anti-mouse secondary (ie. Goat Anti-Mouse)
Potential Cause: Storage issues
- Freeze/thaw cycles are detrimental to the antibody structure and can cause degradation. It is best to aliquot antibodies into one time use vials upon delivery
- Antibody was not stored as recommended. Unfortunately this might require a new vial to be used instead
Potential Cause: Antigen level is too low
- Load sufficient protein onto the gel (~20 µg)
- It also may be necessary to induce cells to produce more protein before harvest
- Ensure protein is not degraded
Potential Cause: Protein is not highly expressed in the cells/tissue
- Concentrate your protein lysates by isolating the cellular compartment containing your protein of interest (ie. mitochondria or cellular membrane)
- Try using ECL western blot rather than the colorimetric western blot
Potential Cause: Epitope is masked by the blocker
- Use a different blocker agent
- Optimize the concentration of the blocker
Potential Cause: Protein did not transfer
- Optimize transfer protocol for specific protein
Potential Cause: Secondary antibody tag not visible
- Ensure that the secondary antibody tag has not been exposed to excessive light
- Use a longer incubation time
Potential Cause: Excessive Washing
- Decrease the number of washing steps
2. Problem: High Background Staining
Potential Cause: High Antibody Concentration
- Reduce the concentration of primary and/or secondary antibody
- Always optimize both primary and secondary antibodies with every experiment
Potential Cause: Insufficient Blocking
- Increase blocker incubation time and/or temperature
- Increase concentration of blocker
- Try a different blocker such as BSA, casein, or milk
- We recommend 5% w/v non-fat dry milk in Tris buffered saline with 0.05% TritonX100, TBST overnight at 4oC or 1 hour at RT
Potential Cause: Insufficient Washing
- Increase the volume and number of washes
- Add or increase concentration of detergent in wash buffer (ie. 0.05% Tween-20)
Potential Cause: Contaminated Solutions
- Be aware of potential contaminants. Always use clean equipment, fresh solutions and wear gloves
Potential Cause: Detection of the blocker by the primary/secondary antibodies:
- Add a mild detergent (Tween 20) to the incubation and washing buffer. For phospho-specific antibodies, always use BSA rather than milk. Milk contains casein which is a phospho-protein
Potential Cause: Overexposure of Membrane
- Reduce exposure times
Potential Cause: Membrane dried out
- Ensure membranes are thoroughly covered in buffer at all times
3. Problem: Non-Specific Bands
Potential Cause: Antibody concentration is too high
- Decrease the concentration of the primary antibody, and run a secondary antibody control
- Refer back to the product datasheet/product page for recommended starting dilutions
Potential Cause: Protein is overloaded
- Load less protein into each well
Potential Cause: Unpurified antibodies can produce non-specific bands
- Try switching to an affinity purified product instead.
Potential Cause: Nonspecific sites were not blocked
- Increase the concentration of the blocker from 5% to 7%
- Increase the blocker incubation time
- Increase the blocker incubation temperature
- Add blocker to antibody dilution buffers
4. Problem: Band size is smaller than expected
Potential Cause: Protein sample is degraded
- Ensure that there is sufficient protease inhibitors in the sample buffer
- Use a fresh sample and keep on ice
5. Problem: Band size is larger than expected
Potential Cause: Protein may form multimers.
- Try boiling in Laemmli buffer for extra time to break down the quaternary structure
Potential Cause: The protein sample has multiple modified forms such (acetylation, methylation, phosphorylation etc.)
- Use a modifying agent to remove post-translational modifications, or review the literature for expected band size of modified forms
6. Problem: Smile effect
Potential Cause: Migration of protein was too fast due to low resistance
- Decrease the voltage while running your gel
Potential Cause: Migration was too hot
- Run the gel immersed in ice-cold buffer, on ice, or in a cold space
7. Problem: Band ran too far, or not far enough
Potential Cause: Proteins were not adequately separated
- Change the percentage of the gel, either by increasing it for smaller proteins, or by decreasing it for larger proteins
- Change the run time of the gel, either by increasing it for larger proteins, or decreasing it for smaller proteins
8. Problem: Patchy background or Black Dots
Potential Cause: Uneven transfer of proteins to membrane
- Roll out any air bubbles between the gel and the membrane to ensure even transfer of proteins
Potential Cause: Secondary antibody aggregates
- Spin down the secondary antibody or remove aggregates through filtration
Potential Cause: Antibodies are binding to the blocking agent
- Filter the blocker to remove aggregates and contamination
Potential Cause: Uneven coverage of membrane during incubation
- Use a shaker to ensure that the membrane is evenly covered with solution
🧪 New Scientifically Relevant Resources (2020–2025)
- Bio-Rad’s Western Blot Doctor™ (2023)
Offers a self-guided diagnostic tool for troubleshooting issues like ghost bands, uneven signal, and blotchy backgrounds.
Visit Bio-Rad’s Western Blot Doctor - MachineSolved’s Quick Fixes & Tips (2024)
Emphasizes sample preparation, blocking buffer optimization, and gel percentage adjustments for better band resolution.
Visit MachineSolved - Thermo Fisher Scientific Troubleshooting Hub
Provides visual guides to identify problems like high background, multiple bands, and uneven staining.
Visit Thermo Fisher’s Troubleshooting Hub
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